Sep 13, 2016 - S12) reveal that PPAP2A is significantly increased (p-value = 0.02; n = 3 females per genotype) in the p53d/d implantation sites. The previ-.
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OPEN
received: 17 May 2016 accepted: 11 August 2016 Published: 13 September 2016
Trp53 deficient mice predisposed to preterm birth display regionspecific lipid alterations at the embryo implantation site Ingela Lanekoff1,2, Jeeyeon Cha3,†, Jennifer E. Kyle4, Sudhansu K. Dey3, Julia Laskin1 & Kristin E. Burnum-Johnson4 Here we demonstrate that conditional deletion of mouse uterine Trp53 (p53d/d), molecularly linked to mTORC1 activation and causally linked to premature uterine senescence and preterm birth, results in aberrant lipid signatures within the heterogeneous cell types of embryo implantation sites on day 8 of pregnancy. In situ nanospray desorption electrospray ionization mass spectrometry imaging (nanoDESI MSI) was used to characterize the molecular speciation of free fatty acids, monoacylglycerol species, unmodified and oxidized phosphatidylcholine (PC/Ox-PC), and diacylglycerol (DG) species within implantation sites of p53d/d mice and floxed littermates. Implantation sites from p53d/d mice exhibited distinct spatially resolved changes demonstrating accumulation of DG species, depletion of Ox-PC species, and increase in species with more unsaturated acyl chains, including arachidonic and docosahexaenoic acid. Understanding abnormal changes in the abundance and localization of individual lipid species early in the progression to premature birth is an important step toward discovering novel targets for treatments and diagnosis. Preterm birth and prematurity are global issues with immediate and long-term social and economic consequences. Thirteen million premature births and more than three million stillbirths occur each year. Prematurity is a direct cause of 35% of all neonatal deaths annually, totaling more than one million deaths worldwide1. Furthermore, many babies who survive premature birth suffer from serious long-term disabilities such as neurodevelopmental impairments, behavioral problems, and respiratory illnesses2. Mice with conditional deletion of uterine Trp53 (p53d/d) serve as a powerful model to study the molecular landscape associated with spontaneous preterm labor3–5. Whereas all floxed (p53f/f) mice experience labor between days 19 and 20 of pregnancy with full complement of offspring, more than 50% of p53d/d mice lacking uterine p53 deliver preterm on days 17 or 18 and lose 100% of the offspring through stillbirth or neonatal death3. We have previously shown that early aberrations during decidualization (e.g., premature decidual senescence) can result in adverse pregnancy outcomes, such as preterm birth3. The process of decidualization is maximal on day 8 of pregnancy to accommodate and support the developing embryo before establishment of a functional placenta, which forms at the mesometrial (M) pole of the uterus. Mice with conditional uterine deletion of p53 have premature decidual growth restriction with polyploidy, accelerated terminal differentiation and decidual senescence on day 8 of pregnancy3. We previously reported markedly enhanced levels of cyclooxygenase 2 (COX-2), prostaglandin F synthase (PGFS) and prostaglandin F2α (PGF2α) on day 16 of pregnancy3. Prostaglandin (PG) species are converted from arachidonic acid, a free fatty acid originating from the metabolism of a variety of lipid species by cyclooxygenases (COX). Comparable signatures of decidual senescence with increased COX signaling were observed in women undergoing preterm birth, making lipid metabolism and signaling clinically relevant5.
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Physical Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. 2Department of Chemistry-BMC, Uppsala University, Sweden. 3Division of Reproductive Sciences, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA. 4Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. †Present address: Department of Medicine, Vanderbilt University, Nashville, TN, USA. Correspondence and requests for materials should be addressed to K.E.B.-J. (email: kristin.burnum-johnson@pnnl. gov) Scientific Reports | 6:33023 | DOI: 10.1038/srep33023
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www.nature.com/scientificreports/ The tumor suppressor p53 maintains genomic stability by triggering cell cycle arrest, DNA repair or apoptosis in response to cellular stresses such as DNA damage, in addition to broader cellular functions. Further, p53 modulates lipid metabolism6 and COX signaling is increased in mouse uterine tissue deficient of p53 expression3. We have previously reported proteomic comparisons of decidua from p53f/f and p53d/d implantation sites on day 8, revealing that Trp53 deficiency negatively affects antioxidant status and ATP production due to mitochondrial dysfunction7. Additionally, we showed a decrease of enzymes in the β-oxidation pathway, the process by which fatty acids are metabolized in the mitochondria7. To investigate regional lipid alterations of p53d/d implantation sites on day 8 of pregnancy, we used nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI)8–10. We have previously used nano-DESI MSI for three-dimensional and MS/MS imaging of lipids and metabolites in mouse embryo implantation sites on day 6 of pregnancy11,12. The data acquired with nano-DESI MSI contains information about hundreds of molecules detected at each predefined x and y coordinate across the sample. Consequently, any detected molecule can be visualized as an ion image, depicting its distribution and relative abundance on the tissue section. Moreover, nano-DESI MSI enables quantification and generation of ion images free of matrix effects by use of internal standards to the extraction solvent13–16. Herein, we employed nano-DESI MSI for examining molecular signatures of preterm birth by comparing the localization and abundance of lipids and lipid metabolites in uterine tissue sections of p53f/f and p53d/d mice. We report significant cell-type specific differences in the abundance of diacylglycerol (DG) and oxidized phosphatidylcholine (Ox-PC) species. The significant alterations in DG and Ox-PC abundances between control and p53 knockout mice indicate that Trp53 deficiency is associated with a severely altered lipid metabolism at an early stage of pregnancy.
Results
Lipid distributions in embryo implantation sites of mice on day 8 of pregnancy were characterized using nano-DESI MSI. At this early stage of pregnancy decidual cell growth and terminal cell differentiation are maximal. Lipid alterations at this sensitive stage of pregnancy can lead to suboptimal pregnancy outcomes3,17. The two most distinguishable microenvironments of the implantation site encompass the decidual cells at the AM-pole, where the embryo first implants, and the M-pole, the site of placental development. The decidual cells in the AM-pole will eventually undergo apoptosis to leave room for the growing embryo while the cells in the M-pole will develop into the placenta as the embryo requires nutrition to grow.
Acyl chain composition is significant for Phosphatidylcholine (PC) localization in implantation sites.
Figure 1A shows ion images of nine abundant phosphatidylcholine (PC) species, acquired by nano-DESI MSI. The 12-μm thick tissue sections from the central part of the p53d/d (top row) and p53f/f (bottom row) implantation sites containing the embryo were collected on day 8 of pregnancy. The color intensity of the ion images depicts the localization of different PC species to distinct cellular regions of each implantation site18. The localizations are highly specific, the difference of a single double bond can restrict the localization of a species to either the mesometrial (M-pole) or antimesometrial (AM-pole) hemisphere of the uterus. For example, PC 36:3 (18:1–18:2) is primarily localized to the M-pole while PC 36:2 (18:0–18:2), with one less double bond, is predominantly localized to the opposite hemisphere of the implantation site, the AM-pole (Fig. 1A). PC 36:1 (18:0–18:1), having only one double bond, is also localized to the M-pole. This distinct distribution of PC species, all of which contain 36 carbons and only differ by having one, two or three double bonds, exemplifies how extremely small variations in lipid structure influence the species-specific localization to unique microenvironments in the tissue. The cellular regions of the AM-pole and the M-pole constitute small subregions of the implantation site tissue sections which only measure ~6 × 4 mm in this study; bulk analysis techniques requiring homogenization would obscure detailed information on the spatial localization of individual lipid species across this complex tissue composed of multiple distinct cell types. However, by use of nano-DESI MSI, regions can be defined post analysis to extract data from specific cellular regions of interest (ROIs) without increasing complexity and uncertainty of the analysis. Mass spectral abundance data was extracted from the defined regions of the M-pole and AM-pole and statistically compared between mouse implantation sites collected from three p53f/f and three p53d/d mice. The ROI analysis revealed that specific PC species displayed a regional relative change in abundance. In Fig. 1B the abundance of individual PC species within the total PC pool is plotted with the M-pole on top and the AM-pole on bottom for the p53f/f and p53d/d implantation sites. PC species with longer and more unsaturated acyl chains exhibited relatively higher abundances within the PC pool of the p53d/d mice. Specifically, PC 40:8, PC 40:4, PC 42:7, and PC 42:6 are relatively increased in the M-pole and PC 36:4, PC 36:5, and PC 42:6 are relatively increased in the AM-pole of the p53d/d implantation sites. Interestingly, all of these PC species contain several double bonds, potentially impacting the biophysical properties of the cellular membranes thereby disturbing homeostasis both on the microscale and macroscale level. Microenvironments affected in single cells and across cellular regions of the uterus at day 8 of pregnancy play a role in preterm labor.
Diacylglycerol (DG) species accumulate in p53d/d implantation sites. Figure 2A displays ion images
of five abundant DG species in implantation sites of p53f/f (bottom row) and p53d/d (top row) and Fig. 2B,C contain the mass spectral intensity data of several DG species extracted from the defined regions of the M-pole (Fig. 2B) and the AM-pole (Fig. 2C). From Fig. 2B,C it is clear that several DG species significantly accumulate in both the M-pole and the AM-pole of the p53d/d implantation sites. Figure 2C further shows that DG species in the AM-pole, the site of embryo implantation, have more polyunsaturated fatty acids (PUFAs) in their acyl chains than DG species in the M-pole. DG species play important and diverse roles as both precursors of phospholipids and as second messengers in intracellular communication events. The accumulation of DG will therefore significantly alter cellular events in these regions. Accumulation of neutral lipids including DG has been previously reported after loss of p53 in both mouse embryonic fibroblast cells and mouse liver6. Scientific Reports | 6:33023 | DOI: 10.1038/srep33023
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Figure 1. Distributions of PC in mouse embryo implantation sites. (A) Optical image and ion images depicting the abundance and localization of abundant PC species in p53f/f and p53d/d implantation sites on day 8 of pregnancy; M, uterine mesometrial pole; AM, uterine anti-mesometrial pole; PC, phosphatidylcholine; the embryo is located in the center of the implantation sites; myometrium (muscle layer) encircles the uterus. The intensity scale bar dark to bright represents low to high intensity. Note that the ion images have individual intensity bars ranging between 0–100%, and, therefore, the intensity colors cannot be compared between two images. Scale bar on optical images shows 1 mm. (B) The relative abundance of PC species within the total PC pool localized to the M-pole (top row) or the AM-pole (bottom row) in the p53f/f (dark gray) and p53d/d (light gray) implantation sites. Significance calculated using the Student’s t-test is depicted with * for p-values
